Herein we report the sophisticated process of structural optimization toward a previously disclosed Src inhibitor,compound 1,which showed high potency in the treatment of triple negative breast cancer(TNBC)both in vitro and in vivo but had considerable toxicity.A series of 3-(phenylethynyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine derivatives were synthesized.In vitro cell-based phenotypic screening together with in vivo assays and structure-activity relationship(SAR)studies finally led to the discovery of N-(3-((4-amino-1-(trans-4-hydroxycyclohexyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-4-methyl-3-(trifluoromethyl)benzamide(13an).13an is a multikinase inhibitor,which potently inhibited Src(IC50 = 0.003 μM),KDR(IC50 = 0.032 μM),and several kinases involved in the MAPK signal transduction.This compound showed potent anti-TNBC activities both in vitro and in vivo,and good pharmacokinetic properties and low toxicity.Mechanisms of action of anti-TNBC were also investigated.Collectively,the data obtained in this study indicate that 13an could be a promising drug candidate for the treatment of TNBC and hence merits further studies.